Enhanced bonding layers on titanium materials

ABSTRACT

The present invention provides a dense-coverage, adherent phosphorous-based coating on the native oxide surface of a material. Disclosed phosphorous-based coatings include phosphate and organo-phosphonate coatings. The present invention also provides further derivatization of the phosphorous-based coatings to yield dense surface coverage of chemically reactive coatings and osetoblast adhesion-promoting and proliferation-promoting coatings on the native oxide surface of a titanium material.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application claims the priority of U.S. ProvisionalApplication Ser. No. 60/300,144, filed Jun. 22, 2001. The presentapplication also is a Continuation-In-Part of U.S. application Ser. No.09/668,080, filed Sep. 22, 2000, which application, in turn, claimspriority from U.S. Provisional Patent Application Ser. No. 60/155,398filed Sep. 22, 1999, and which is also a Continuation-In-Part and aDivisional of U.S. patent application Ser. No. 08/794,833, filed Feb. 4,1997, which application, in turn, claims priority from U.S. ProvisionalPatent Application Ser. Nos. 60/028,949 filed Oct. 17, 1996 and60/035,040 filed Jan. 13, 1997. The disclosures of all six applicationsare incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to coatings bonded to the oxide surface oftitanium metal and titanium alloys (collectively, titanium materials) bytreatment of the oxide surface with phosphorous-based acids. The presentinvention further relates to implantable medical devices fabricated fromtitanium materials having these bonded coatings which impart anosteoconductive surface to the medical device. In addition, the presentinvention relates to methods for forming such coatings on the surface ofimplantable medical devices to provide an osteoconductive surface, andmethods for using implantable devices bearing an osteoconductivesurface.

BACKGROUND ART

Titanium metal combines the strength of steel with the light weight ofaluminum, and for this reason the metal and its alloys are usedextensively in the aerospace and aviation and high performance sportsequipment industries. Titanium metal and its alloys (hereinafter,“titanium materials”) are also resistant to corrosion under ambientconditions. Because of its mechanical properties and chemical resistanceto degradation by bodily fluids, titanium materials are used extensivelyto fashion metal fitments used as dental and orthopedic implants(medical implants). When a metal fitment is used as a medical implant,it is important to create a stable bond between bone tissue and thesurface of the implant. Poor bonding at the interface between thesurface of the implant and the bone tissue leads to low mechanicalstrength of the bone-to-implant junction and the possibility ofsubsequent implant failure. An important goal for interface optimizationis to use species which are biocompatible and which enable bonemineralization at the interface following implantation. Bone tissue is acombination of protein and mineral content, with the mineral contentbeing in the form of hydroxyapatite. Currently, there is no effectiveway to obtain strong attachment of incipient bone with the implantmaterial at the interface between the surfaces of the two materials inorder to “stabilize” the implant.

The problem of synthesizing an adhesion-promoting interface on implantsis often approached from the prospective of high temperature methods,including using plasma or laser-induced coating techniques. However,these methods engender problems of implant heating and surface coverage.For example, calcium phosphate deposition at high temperatures can giverise to ion migration. Plasma-induced phosphate coating of a titaniumsubstrate gives surface hydroxyapatite as well as surface calciumphosphate, titanates and zirconates. Therefore, control of surfacestoichiometry can be problematic, and defects at the interface maytranslate into poor mechanical strength.

The use of intermediate layers, for example of zirconium dioxide, toenhance hydroxyapatite adhesion and interface mechanical strength hasbeen explored with success. However, a practical limitation involvinglaser or plasma deposition is that it is hard to obtain comprehensivecoverage on a titanium implant of complex 3-dimensional structure. Thezirconium dioxide interface formed at high temperatures is of lowsurface area and maintains few, if any, reactive functional groups forfurther surface modification chemistry. A silicate, phosphate orphosphonate interface can function to nucleate the growth ofhydroxyapatite, thereby minimizing implant failure and the attendantneed for serial revision implant surgery, which can be a consequence ofunstable implant-to-bone interaction, but, as described below, there islittle success of providing such surfaces on the oxide surfaces oftitanium materials.

Solution-phase surface processing does not suffer from the practicallimitations of surface coverage that can be attendant with plasma orlaser-based deposition methods, and procedures involving formation ofhydroxyapatite from solution, often using sol-gel type processing, havebeen accomplished. Elegant methodologies have been developed in whichgraded interfaces have been prepared, extending from the pure implantmetal to the biomaterial at the outer extremity by way of silicates.However, while solution-based procedures are inexpensive and give riseto materials resistant to dissolution by bodily fluids, adhesion of thehydroxyapatite to the implant metal is less strong than is observed whendeposition is accomplished by plasma spraying techniques.

The deficiency of these solution approaches may lie in the nature of thenative oxide surface of titanium materials. Exposure of a clean surfaceof titanium materials to oxygen results in the spontaneous formation ofsurface titanium oxides (native oxide). The exact chemical stoichiometryand structure of these oxides varies from material to material, and withdepth in the oxide layer, with environmental variables, and with theprocessing history of the material. The oxide layer may bestoichiometric, super-stoichiometric, or sub-stoichiometric with respectto TiO₂, a stable oxide of titanium. Generally, the uppermost layer ofthe native oxides comprises some form of TiO₂. It may be crystalline,but if crystalline, it is generally disordered. Typically, manydifferent phases exist within the oxide layer between the metal and theambient environment. Generally, the uppermost layer of oxide includeswidely dispersed hydroxyl functional groups bonded to a titanium atom.The surface forms spontaneously by exposing the metal or alloy to theambient environment, and is alternatively described as the “native oxidesurface” of a titanium material.

This oxide layer protects the underlying material from further chemicalattack in that it is generally resistant to further chemical reaction.This property of the oxide surface of titanium materials mitigatesagainst the adhesion other moieties to it, for example, bone tissue, andalso makes application of an adhesion-promoting coating to the nativeoxide surface problematic. For example, a phosphorous-based acidincorporating into its structure an organic moiety, for exampleorgano-phosphonic acid, does not readily form an adherent coating withthe bulk metal under ambient conditions. This is in contrast to othermetals that possess oxide coatings, for example, tin, iron, aluminum andcopper, or their alloys, for example, steel, or bulk oxides, forexample, mica, all of which yield an adherent film when treated withsuch acids. Film formation of the type observed between, for example,zirconium surface oxides and organophosphonic acids, is not observedwith the oxide surface of titanium materials. An example of this isdescribed by Gao et al. in Langmuir, Vol. 12 (1996) p. 6429.

For many materials other than titanium, as described above, the nativeoxide surface can be altered chemically (derivatized) by exposing thesurface to various hydrolytically reactive reagents. An example ofderivatizing an oxide surface in this manner is exposure of the surfaceof a silicate (silicon oxide) to a trimethylsilyl derivative which“silanates” the surface, converting it from a hydrophilic surface to onethat is hydrophobic by bonding trimethyl silyl functional groups to thenative oxide surface. Typically, chemically derivatizing a surface isthe most cost-effective method to achieve uniform surface coverage onsurfaces having complex shapes, and in general represents the leastimpact to the mechanical properties of the material derivatized comparedto other surface modification techniques.

Silanization has long been considered the benchmark method for attachingorganics to titanium and its alloys, for example, Ti-6Al-4V, via theirnative oxide coatings. Direct silane-surface bonding is limited bysurface hydroxyl group content, and the OH group content of the Tinative oxide surface is low, accounting for only about 15% of totalsurface oxygen. Low yields of direct surface silanization result, andsilane reagent crosslinking can be the dominant mode of reactivity.Unfortunately, surface-bound and cross-linked siloxanes can behydrolytically unstable, which can further reduce amounts of keyorganics that are coupled to simple surface silanization reagents underaqueous conditions.

It will be appreciated that for metal oxide surfaces which are notderived from titanium metal or its alloys, for example, the oxidesurface of aluminum and its alloys or the oxide surface of silicon, itis common to carry out reactions between organometallic complexes andhydroxyl groups terminating the oxide layer to provide for a surfacewhich is amenable to further derivatization or passivation. For these“non-titanium material” oxide surfaces, in cases in which the naturaloccurrence of hydroxyl groups per unit area of surface is too sparse toprovide for the formation of a dense surface layer of the derivatizationproducts, it is known to subject the oxide surface to a variety ofhydrothermal treatment schemes to increase the density of hydroxyl sitesand improve the reactivity of the surface toward derivatization.

In general, the coverage of naturally occurring hydroxyl sites whichform on titanium material oxide surfaces is too sparse to to providedense-coverage layers of derivatization product on the surface of thematerial by traditional derivatization routes. Additionally, attempts toincrease the actual density (sites/unit area) of hydroxyl sites in thesurface oxides of titanium materials results in “roughening” of thesurface, which, while increasing the number of hydroxyl sites projected(nominal) surface area of the bulk material, also increases the actualsurface area of the material, and, consequently the density of hydroxylsites/unit area remains approximately constant.

If the protective oxide layer of a titanium material is dissolved underconditions in which its re-formation is inhibited, rapid corrosion ofthe material will occur, an example of which is reported by Fang et al.in Corrosion, Vol. 47, (1991), p 169. Reducing acids, for examplehydrobromic, sulfuric, and phosphoric acid, under the proper conditionsof heat and acid concentration, can attack titanium metal and itsalloys. Such attack is especially facile when oxidizing agents, forexample, air, are excluded from the surface of the material underattack. For example, titanium metal rapidly dissolves in 85% phosphoricacid at 80-100° C. yielding a solution from whichtitanium(III)dihydrogen orthophosphate (Ti[H₂PO₄]₃, (hereinafter,“Ti-phosphate”) can be isolated.

A need exists for a methodology that combines the benefits such as thoseobtained from the physical deposition of interfacial zirconium dioxidewith the coverage, processing and speciation control of solution-basedmethods described above. Additionally, a need exist for a methodologythat makes the oxide surface of titanium materials amenable to solutionprocess derivatization that establishes dense-coverage, robust andadherent coatings amenable to establishing a strong interface betweenthe oxide surface and materials with which the surface is placed incontact, for example, adhesion between medical implants made of titaniummaterials and the tissues into which they are implanted.

SUMMARY OF THE INVENTION

It has now been discovered that heating a self-assembled layer of aphosphoric or phosponic acid on the native oxide surface of a substratewill form an adherent coating layer that may be further derivatized withadherent species. Therefore, according to one aspect of the presentinvention there is provided an adherent, self-assembledphosphorous-based coating layer bonded to the native oxide surface of amaterial by heating a self-assembled layer of a phosphorous-based acidformed on the surface of the oxide until the self-assembled layer isbonded to the oxide surface, the phosphorous-based acid being chosenfrom phosphoric- or organo-phosphonic acids.

The present invention thus includes the methods by which the coatedsubstrates of the present invention are formed. Therefore, in accordancewith another embodiment of the present invention there is provided amethod of bonding a layer of a phosphorous-based acid moiety to atitanium oxide surface comprising coating said oxide surface with aphosphorous-based acid moiety self-assembled layer and heating saidcoated oxide surface until said self-assembled layer is bonded thereto,the phosphorous-based acid moiety comprising the self-assembled layerbeing selected from the group consisting of phosphoric acid andorgano-phosphonic acids.

Preferred coatings are those which have been formed from phosphoric acidand alkylene- and arylene-organo-phosphonic acids, including substitutedalkylene and arylene-phosphonic acids. More preferred are substitutedalkylene phosphonic acids with a reactive substituent omega to thephosphonic acid functional group. Preferred oxide surfaces are thenative oxide surfaces of titanium materials. It is preferred for thephosphoric acid to be in the form of an aqueous solution having a pHmore acidic than about pH 3.0.

The coatings of the present invention promote the adhesion of bonetissue to the substrates on which they are coated. Therefore, inaddition to the coatings of the present invention and the methods bywhich they are formed, the present invention also provides methods forcoating implantable medical devices having a titanium oxide surface forcontacting bone tissue with the coating layers of the present invention,coated implantable devices and implantation methods incorporating thecoated devices.

Other features of the present invention will be pointed out in thefollowing description and claims, which disclose, by way of example, theprinciples of the invention and the best methods which have beenpresently contemplated for carrying them out.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention provides a dense-coverage, adherent,phosphorous-based coating bonded to the native oxide surface of atitanium material. The present invention also provides modifiedphosphorous-based coatings that effectively bond a dense-coverage,adherent coating of chemical species to the native oxide surface of atitanium material. Thus, the present invention provides, under mildconditions, on a native oxide surface of a titanium material, aderivitized surface which modifies the chemical properties of the nativeoxide surface. The present invention also provides a method of producingthose surfaces which may be adapted to provide an even coating of thederivatized surface over a complex shape.

While the present invention can provide derivatized surfaces on thenative oxide of any titanium material, it is anticipated that thepresent invention will be most useful in the provision of derivatizedsurfaces which promote bone adhesion to the surface of medical implants,thus the present invention also provides a derivatized surface thatpromotes the adhesion and proliferation of osteoblasts, and provides amethod of synthesizing such a surface. This aspect of the presentinvention is particularly useful for securely bonding replacement jointsto bone tissue, for example, knee and hip replacements. Therefore, inaddition to the coatings of the present invention and the methods forforming the coatings, the present invention provides a coating forimplantable medical devices, methods for improving the adhesion to bonetissue of implantable medical devices, and methods for implantingmedical devices by first coating them according to the presentinvention.

The coatings of the present invention are more amenable to chemicalinteraction with other chemical species than the native oxide surfaceupon which they are formed, but the underlying material (substrate)retains some of the native oxide surface, and thus thedegradation-resistant properties of the native oxide surface. It will beappreciated that the coatings of the present invention may be formed aswell on the native oxide surfaces of materials other than titaniummaterials, and will be equally effective in providing a surface havingdense-coverage by moieties which are amenable to the furtherderivatization reactions, for example, those described below.

The phosphorous-based coating of the present invention is adense-coverage, adherent phosphate or phosphonate layer bonded to thenative oxide surface of a substrate of titanium material. As describedabove, these layers can promote adhesion between the titanium materialand an applied chemical species, for example, when applied to a medicalimplant which is then contacted to living bone tissue, the layer canpromote adhesion between the implant upon which such surface layers areformed and bone tissue. The present invention also provides adense-coverage, adherent, derivatized surface on the oxide coating of atitanium material which is amenable to further derivatizing reactionsthat utilize hydrolytically reactive reagents to thereby provide adense, adherent surface coating of these derivatizing species.

The methods of producing the phosphorous-based coatings of the presentinvention are not critical. It will be appreciated that many variationsmay be employed. In general, the surface to be coated is contacted witha phosphorous-based acid solution, the solvent removed, and the surfaceto be coated is then heated and held at temperature for a period of timewhile reaction between the phosphourous-based acid and the oxide surfaceproceeds.

In a typical coating procedure, the surface to be coated is prepared bysanding the surface clean, rinsing the sanded surface with solvent, forexample, methanol, methylene chloride, and methyl-ethyl ketone, anddrying the surface under conditions which preclude physisorbed water,for example, at 200° C. in the ambient environment.

Next, the surface is exposed to a phosphorous-based acid solution, andthe solvent is allowed to evaporate, leaving a self-assembled film ofphosphorous-based acid moieties on the surface.

Next, reaction between the film of phosphorous-based acid and thesurface is initiated, for example, by heating the surface, for example,by placing the treated surface and substrate in an oven.

It will be appreciated that other methods of providing a coating ofphosphorous-based acid on the oxide surface may also be employed, forexample, by dipping the surface to be coated into a vessel containingthe acid solution and draining the excess. A variation of this exampleis to dip the surface into a heated solution of the acid for a period oftime which initiates reaction but does not substantially corrode thematerial, followed by annealing the material in an oven.

The coatings of the present invention are stable. Once formed, so longas fresh acid is not present, continued heating of the substrate andsurface do not result in further reaction or degradation of the nativeoxide or phosphorous coating.

Typically, concentration of acid solutions will range from about 0.01 toabout 30 mM of the acid species, although concentrations lying outsideof this range may be used, for example, when the reaction rate of thephosphorous-based acid chosen is low.

The layer forms spontaneously and may be formed at any substratetemperature. Typically, it will be formed at temperatures between about20° C. and about 200° C. to permit uniform application of the acidspecies to the surface oxide to be coated. Lower or higher temperaturesdepending on time to form and solubility of acid species. Temperaturesoutside of this range may be employed where solubility and/or reactionsrates of the chosen acid species do not preclude doing so.

The reaction forming the phosphorous-based coating of the presentinvention (also described as “annealing”) is typically carried out inthe ambient environment because it is usually convenient to do so. Itwill be appreciated that it may also be carried out in inertenvironment, for example, in a vacuum oven or in an inert gas glow box.

Typically, the oxide surface is annealed at temperatures between about100° C. and about 200° C. because the rate of film formation isreasonable at these temperatures and such an environment is accessibleusing simple ovens. Higher or lower temperatures may also be employed.

In general, it is convenient to drive the reaction using hot air heatingof the surface, for example, using a drying oven, but methods ofimparting energy to the surface, for example, radiant, microwave, orlaser radiation, may also be used.

It will be appreciated that numerous means may be employed to evaporatethe solvent and to drive the reaction of the phosphorous-based acid withthe surface that will also provide the phosphorous-based coatings of thepresent invention.

Substrates suitable for use with the present invention include anytitanium metal or alloy (hereinafter, “titanium materials”) thereofcapable of forming a native oxide surface. As described above,implantable medical and dental devices (hereinafter, “medicalimplants”), are frequently made from titanium materials.

Titanium metal from which medical implants are made typically has apurity such that the mass of the material is greater than about 98 wt. %titanium, for example, the Allvac series of titainium metal availablefrom Allegheny Technologies Company, for example, Allvac 70, whichcomprises about 98 wt. % titanium and Allvac 30 CP-1, which comprises99.5 wt % titanium. Although the present invention is applicable to bulktitanium metal which is of either lower or higher purity, and suchmaterial is not outside of the scope of the present invention, ingeneral the lower purity material is not employed as medical implantmaterial.

Typical titanium alloys from which medical implants are made contain atleast about 80 wt % titanium, with the remainder comprising other metalsand trace elements. Examples of titanium alloys (titanium materialscontaining more than trace amounts of other metals) which are used inthe construction of medical implants are also found in the Allvac seriesof titanium alloys available from Allegheny Technologies Company, forexample, Allvac Ti-15-Mo, which contains about 15 wt. % Mo and in excessof about 84% titanium, Allvac 6-4, which contains about 4% vanadium,about 6 wt. % aluminum, and in excess of 89 wt % titanium (which alloyis also described herein as Ti-6Al-4V), and Allvac 6-2-4-Si, whichcontains about 6 wt % aluminium, about 2 wt % molybdenum, about 2 wt. %tin, about 4 wt. % zirconium, and in excess of 85 wt. % titanium. Otherpurities and specifications of titanium alloys, whether used for theconstruction of medical implants or not, are known, and are alsoamenable to the present invention, and are therefore also contemplatedin the term “titanium materials”.

For the application of phosphonate coatings using the general proceduresdescribed above, the phosphorous-based acid used is selected from theorganic phosphonic acids. For purposes of the present invention,“phosphonic acid” refers to compounds having the formula H₂RPO₃, whereinR is an organic ligand with a carbon atom directly bonded to phosphorus.

Phosphonic acid species which are useful in the formation of coatings ofthe present invention may have, as the organic ligand of the molecule, ahydrocarbon which comprises an alkylene or arylene. Generally, usefulalkylene and arylene hydrocarbon ligands will comprise between about 2and about 40 carbon atoms, although the present invention contemplatesorganic portions outside of this range as the properties desired of thecoating formed dictate larger or smaller organic portions.

An alkylene organic ligand of a phosphonic acid suitable for use in thepresent invention may be linear or branched, saturated or unsaturated,and unsubstituted or with one or more substituents. An arylene organicligand may comprise direct attachment of an aromatic moiety to thephosphorous atom of the phosphonic group, or it may be attached by anintervening alkylene moiety. Additionally, the arylene ligand may beincorporated into an alkylene chain (an arylene moeity having two ormore alkyl substituents) or be a substituent depending from an alkylenechain. Substituent from arylene moieties may additionally beunsubstituted or may have one or more additional substituents.

Substituents on the hydrocarbon portion of phosphonic acids useful inthe present invention may be appended to any carbon atom of thehydrocarbon ligand. Useful substituents are, for example, reactivefunctional groups, for example, a hydroxyl group, carboxylic group, anamino group, a thiol group and chemical derivatives thereof. It will beappreciated that any functional group which can participate in a furtherderivatization reaction can be employed. Additionally, an alkylenehydrocarbon ligand may contain within the structure or appended to thestructure, reactive moieties, for example sites of unsaturation, whichmay be further reacted in a polymerization reaction with reactivesubstituents on the hydrocarbon ligands appended to other phosphonatesites bound to the surface of the native oxide during a phosphonatederivatizing reaction. In this manner, a phosphonate-organo-polymericlayer may be formed on the oxide surface. An example of such apolymerization reaction is the preparation of a surface coating ofacrylic phosphonic acid. Unexpectedly, when acrylic acid and methacrylicacid substituents are employed, the polymerization proceedsspontaneously upon exposure to air. For less reactive coatings, thepolymerization can be performed by exposing the coating to conventionalpolymerization reagents and conditions.

In a particularly preferred embodiment, coatings are formed fromphosphonic acids having an organic ligand functionalized at theomega-carbon of the ligand which is further reacted to form covalentbonds with chemical precursors of bone tissue protein, such as aminoacids, or with the bone tissue protein itself. For omega-functionalizedphosphonic acids, the application of the acid to oxide surface generallyresults in a self-assembled phosphonic acid film with the omega-carbondirected away from the substrate surface and available for covalentbonding or further chemical modification. Preferred omega-functionalgroups include hydroxyl, amino, carboxylate and thiol groups.

Another class of substituents which may advantageously be bonded to aphosphonic acid organic ligand are pi-electron delocalized moieties.Essentially any pi-electron delocalized compound capable of reactingwith a transition metal alkoxide to covalently bond a ligand of themoiety to the transition metal is suitable for use with the presentinvention. Particularly useful compounds are pi-electron delocalizedaromatic ring compounds. A particularly preferred aromatic ring compoundis a phenol, which has a relatively acidic hydrogen that is readilytransferred to the transition metal alkoxide to initiate a reaction thatresults in the formation of a transition metal phenolate. Five-memberedheteroaromatic ring compounds having proton-donating ring substituentscapable of reacting with the transition metal alkoxide are alsodesirable because of their high degree of pi-electron delocalization.Examples of such rings include furan, thiophene and pyrrole.

It will also be appreciated that the reactive substituents pendant onthe organic portion of a phosphonate bound to the oxide surface can befurther reacted with reagents which are subject to hydrolysis reactions.Examples include metal alkoxides, examples of which are those having thestructure M(O—R), where M is a metal, R is a linear or branched,saturated or unsaturated, aliphatic or aromatic, substituted orunsubstituted hydrocarbon moiety, and “n” is equal to a stable valancestate of the metal. Examples of metal alkoxide compounds arezirconium-tetra-t-butoxide and silicon-tetra-t-butoxide where R is at-butyl group, M is respectively Zr and Si, and “n” is four. It will beappreciated that other hydrolytically reactive compounds which have twoor more alkoxide ligands in addition to other ligands may also beutilized. In general, alkoxide ligated metals in groups 4 throgh 14 willfind utility in these secondary functionalization reactions withphosphonate coatings of the present development.

Adherent, dense-coverage, phosphate coatings bound to the native oxidesurface of a titanium material (hereinafter, “a coating ofTi-phosphate”) may be prepared by treatment of the surface under mildconditions with phosphoric acid according to the general proceduredescribed above. For purposes of the present invention, “phosphoricacid” is defined according to its' well-understood meaning, H₃PO₄. Inthe process of the present invention, treatment of a native oxidesurface with phosphoric acid forms an inorganic phosphate coating thatis rich in free hydroxyl groups. When the native oxide surface of atitanium material is coated with a phosphate coating of the presentdevelopment and analyzed by XRD, two different titanium phosphatespecies were identified on its surface. One component, Ti₄H₁₁(PO₄)₉.H₂O,could be easily removed by rinsing with water, but the other,Ti-phosphate, remained on the surface. Indeed, XRD analysis of therinsed foil, which had a dull purplish gray color, showed peaks only forTi-phosphate, which were identical to those of powdered H₂TiPO₄. Thereis no long range order to the Ti-phosphate coating, and profilimetry ofthe surface (at 5 mm/s with 5 mg force) showed rough surfaces. Theresistance of Ti-phosphate to removal from Ti by rinsing of “peeling”with Scotch® tape was verified by XRD analysis; the change in relativeintensities of XRD peaks for Ti-phosphate on the Ti substrate weremeasured before and after these tests was inconsequential. Since thereis no preferred orientation for Ti-phosphate on the Ti substrate,phosphate group-derived hydroxyls of Ti-phosphate are likely alsorandomly oriented. The hydroxyl groups of the present inventionphosphate coatings are available for further chemical modification(derivatizing), and may be reacted with, for example, hydrolyticallyreactive reagents, as described above for the phosphonate layers havingreactive substituents. As with the phosphonate coating, further reactionof the phosphate hydroxyl moieties results in dense coverage of thesurface by the derivatizing species. In this manner, species which wouldonly provide a sparse coating on the native oxide if reacted directlycan be used to provide a much denser coating on the phosphatederivatized surface.

Aqueous phosphoric acid solutions having a concentration up to about 3.0M are preferred. For preparation of phosphate coatings of the presentinvention, phosphoric acid solutions having a pH more acidic than aboutpH 3.0 are preferred. Although these preferred ranges are convenient forproviding coatings of the present invention, values outside of thisrange may be employed when reactivity and solubility considerationspermit.

The concentration of phosphonic acid required to form an inorganicphosphate coating on an oxide surface is that concentration ofphosphoric acid effective to form a stable film on the substrate surfacewithout excessively dissolving the substrate. This can readily bedetermined by those of ordinary skill in the art without undueexperimentation.

As with the coatings of phosphonate containing hydroxyl substituents,the hydroxyl groups of the Ti-phosphate coatings of the presentinvention can also serve as reactive sites for covalent attachment ofhydrolytically reactive reagents, for example, Zr or Si alkoxides. It isobserved, by comparison of infrared absorbance by a characteristicfeature of a surface bound moiety, that surface loadings of theseorganometallics are 1-2 orders of magnitude higher on Ti-phosphatecoatings than those obtained on the native oxide of Ti in which onlyabout 15% of surface oxygen is derived form hydroxyl groups.

Alkyl amines and silanes are reagents commonly used to couplefunctionalized organics to a variety of hydroxylated surfaces, and bondreadily to the phosphate surfaces of the present invention. For example,octadecyl(triethoxy)silane reacts irreversibly with Ti-phosphate but notwith the Ti native oxide surface under comparable conditions. Thephosphate surfaces of the present invention may be further derivatizedby reagents typically used to react with hydroxylated oxide surfaces ofnon-titanium materials.

As described above, the native oxide surface of titanium materials isnot amenable to profound alteration of the chemical properties of thesurface using typical derivatizing reactions. In addition, as describedabove, the coverage of hydroxyl groups on a native oxide surface oftitanium materials is sparse, thus, derivatizing reagents which reactwith hydroxyl groups (hydrolytically reactive reagents) typically yielda coverage by the derivatizing species which is too sparse to providefor a significant change in the behavior of the surface of the material.This is particularly problematic with respect to attempts to alter thenative oxide surface of titanium materials with these reagents topromote adhesion when the materials are placed in contact with bonetissue.

The phosphate or phosphonate coatings of the present invention provide alayer which is sufficiently adherent and provides dense-coverage of areactive surface directly bonded to the native oxide surface of titaniummaterials. Studies indicate that coverage yielded by reactioningphosphate coating hydroxyl groups of the present invention withderivatizing reagents yield coverage of the oxide surface that is aboutone to two orders of magnitude greater than that obtainable by directreaction of the derivatizing reagent with the surface hydroxyl groups ofthe native oxide surface. These dense-coverage, adherent phosphate orphosphonate coatings also can promote the adhesion of bone tissue, andare amenable for further derivatization by chemical species whichfurther promote adhesion and proliferation of osteoblasts.

The phosphate coatings of the present invention are rich in freehydroxyl groups. The phosphonate coatings can be made to have hydroxylgroups by using precursor acids having hydroxyl group substituents. Eachof these coating layers may be further functionalized to promotecovalent attachment to bone tissue proteins, or precursors thereof, forexample, by using thiol compounds of the type conventionally employed topromote adhesion between gold metal implants and bone tissue. Thehydrocarbon ligands of the organopolyphosphonate coatings may likewisebe functionalized at a substituent on the organic ligand portion asdescribed above for phosphonate ligand coatings to form covalent bondswith chemical precursors of bone tissue protein or with the bone tissueprotein itself.

The coatings of the present invention can be applied to essentially anyimplant intended for bone or dental tissue contact fabricated from amaterial having an oxide surface at the intended bone or cental tissueinterface. Implants made of titanium and alloys thereof may be employed,as well as implants which are made of materials that can be providedwith an adherent titanium oxide surface. Additionally, thephosphorous-based coatings of the present invention may be applied tooxide surfaces of materials other than titanium-materials and providesimilar potential for bone adhesion.

The methodology of the present invention enables strong adhesion betweena dental or osteopathic implant and incipient bone tissue via a networkof strong chemical bonds. An implant device can be fabricated and itssurface processed ex-situ to provide a composite coating on the implantsurfaces that will give rise to a strong, non-fracturablebone-to-implant seal following implantation. The methodology is amenableto vapor-phase or solution-phase (aerosol spray-on or “dip coating”)chemistry and proceeds under mild conditions, especially compared toplasma or laser-induced deposition. Adhesion of the phosphorous-basedsurface coating has been found to exceed 40 M Pascals of shear stress.

More complex species, for example, a protein or peptide, may also bebonded via the derivatized surface of the present invention to theunderlying native oxide surface of an implant. For example, bonding thefibronectin cell attachment peptide, arginine-glycine-aspartic acid(RGD) to a surface through an organic tether is thought to enhance theosteoconductivity of the surface by providing sites for cell attachmentand spreading. As described above, conventional methods for suchproviding surface peptide attachment to Ti alloys are often problematicand only low yields of such attachment are possible. Using the surfacebonded coating of the present development, for example, acarboxylate-functionalized phosphonate coating, a cysteine-modifiedfibronectin cell attachment peptide (RGDC), which is commerciallyavailable (American Peptide), affords the possibility of attachment ofthe peptide to a reactive site on a surface of the present invention viaformation of a thiol-ester bond using the surface coating of the presentinvention treated with traditional organic derivatization reactiontechiques. It will be appreciated that other derivatization reactionsare also possible.

EXAMPLES

Example films of phosphonates and phosphates were prepared on coupons ofmetal foil or on disks of metal cut from billet. As noted, samples wereprepared in some cases by dip coating the coupon in a bulk solution ofthe coating moiety and in others by aerosol application of the solutionto a surface of the coupon. Aerosol application of monofunctionalphosphonic acids was carried out by dissolving the acid intetrahydrofuran (THF), spraying the acid solution onto the target oxidesurface. As noted, aerosol application was carried out either in theambient environment by spraying a solution of the acid from a pump-spraybottle, or with the target surface residing in a standard nitrogencharged glove box.

The solvent was allowed to evaporate from the sample either with gentleheating and/or a gas current, for example, nitrogen flowing over thesurface, or left to evaporate to the ambient environment by spraying ina solution of the acid from a pump-spray bottle. Where noted, solventevaporation was carried out in the ambient environment, or in an inertatmostphere glove box. For application of difunctional phosphonic acids,two procedures were followed. In the first procedure, a THF solution ofthe phosphonic acid was applied to the target oxide surface while itssubstrate rested in the ambient atmosphere on a hot plate to aidevaporation of the solvent. The treated oxide surface and substrate werethen transferred to a 120° C. oven and annealed at oven temperature forup to 48 hours. Following removal from the oven and cooling, thederivatized surface was rinsed with dry, distilled THF to ensure onlybound species remained. The residue of rinsing solvent remaining on thecoupon was evaporated and the coupon surface was subjected to analysis.

In the second procedure, the substrate was placed in a vessel containinga quantity of acid solution, the solvent was evaporated with thesubstrate in place and the substrate was annealed in an oven to reactthe phosphoric acid solvent to the surface with the native oxide.

In the formation of phosphate coatings, spray or dip procedures,described above, were employed to pre-coat the native oxide surface withphosphoric acid solution. Where noted, phosphoric acid was used aseither a THF or aqueous solution.

Films were analyzed using a quartz microbalance and by FTIRspectroscopy, X-ray powder diffraction spectroscopy, contact anglemeasurement and “peel testing”. The following procedures were used.

Quartz Crystal Microbalance (QCM)

The QCM technique allows accurate, gravimetric determination of masschanges on an electrode which is deposited on a piezoelectric quartzcrystal. It is, thus, ideal to monitor surface reactions of targetmetals when they are used as such electrodes: the QCM oscillates at aresonant frequency which is determined by the cut and mass of thecrystal, and, just as for a classical oscillator, changes in electrodemass result in changes in crystal resonant frequency. Since ourexperiments necessitated detaching active crystals from the QCMoscillator for extended periods of time followed by reattachment,control measurements had to be made of reference crystals which weresubjected to similar handling, but without surface treatment. Up tothree different reference crystals (prepared in different batches) wereused as received to calibrate the QCM. Careful handling of the activeand reference crystals was observed to prevent unacceptably large (>10Hz) frequency change from the initial value, during an experimental run.To ensure that monolayer coverage (at most) occurred on Ti surface,phosphonic acid-based films were subjected to copious rinsing followedby evacuation (≦10⁻² Ton) until a constant crystal frequency wasestablished (within experimental noise levels of ±2 Hz).

Piezoelectric quarts crystals (International Crystal Manufacturers[ICM]; AT-cut, 1000 Å Ti electrodes, 10 MHz, overtone polished, 0.201in. electrode diameter) were used for film deposition and as references.The QCM circuitry was allowed to stabilize for 30 min. after start-up,before experimental measurements were made. In each experimental run,the fundamental frequency (f_(o)) of an unreacted crystal was measured.The crystal was then removed from its holder, aerosol sprayed (on bothelectrodes) with a solution of the phosphorous-based acid and heated at120° C. for 3 days. A new frequency (f_(c)) was then measured. Thecrystal was then subjected to rinsing followed by evacuation (<10⁻² Ton)until a constant frequency was measured (±2 Hz), assumed to be amonolayer coverage of the Ti electrodes. The difference between themonolayer-loaded and the unreacted crystal was then related to theamount of material chemisorbed on the Ti electrode active area.

The quartz crystal microbalance (QCM) was driven by a home-built Clapposcillator and powered by a Hewlett Packard 6234A Dual Output PowerSupply. The frequency of the crystal was measured using a HewlettPackard 5334B Universal Counter and a record of the frequencies wastracked using a laboratory computer. A change in the observed frequencyindicated a change in the mass of the crystal. To insure that all thefrequency changes were attributable to the deposition of the reactants,the frequency of the crystal was monitored before and after exposure toreactants.

X-ray Powder Diffraction

Samples were analyzed by X-ray diffraction using a Rigaku Miniflexspectrometer with CuK α radiation and a Zn filter. Samples were scannedfor 20=8−55° (0.04°/2 sec). Data were analyzed and refined and matchedwith that of known species using Jade 3.0 Pattern Processing forWindows. Samples were placed on glass microscope slides using DowCorning Vacuum Grease, and were placed in an appropriate holder.

Infrared Spectroscopy

Samples were analyzed using either a Nicolet 730 FT-IR equipped with aSpectra Tech diffuse reflectance (DRIFT) attachment or a MIDACIlluminator equipped with a Surface Optics specular reflectance head.When the Nicolet was used for analysis, infrared experiments wereperformed using a glancing angle attachment, a Variable Angle SpecularReflectance Model 500, obtained from Spectra Tech. The angle between thesurface normal and the incident beam was approximately 87°. For bothinstruments, samples were purged with nitrogen for half an hour toreduce the amount of water on the surface, and 1,000 scans werecollected to obtain a reasonable signal to noise ratio. All spectraobtained were ratioed against a spectrum of a clean native oxidesurface.

“Peel-Testing”

Coupons which had been treated were rinsed several times with depositionsolvent and, where appropriate, ethanol and/or water, to remove solubleresidues. A piece of tape (e.g. 3M red Scotch® “650” tape or ScotchMasking Tape #234; 37 oz/in adhesion to steel) was adhered to thederivatized surface of the solvent washed foil sample and quicklyremoved. The freshly “peeled” coupon was then analyzed again by DRIFTspectroscopy.

Contact Angle Measurement

Contact angles were measured at room temperature and ambient conditionson a Tantec Contact Angle Meter CAM-Fl.

All reagents were obtained from Aldrich Chemical unless otherwise noted.Propionic acid (99+ percent), octanoic acid (99.5+ percent), and stearicacid (99.5+ percent) were used as received, 11-phosphonoundecanoic acid(an 11 carbon atom, linear difunctional phosphonic acid with anw-carboxylic acid functional group to the phosphonic acid)11-hydroxy-undecylphosphonic acid (a linear, 11 carbon atom difunctionalphosphonic acid having an w-hydroxyl functional group to the phosphonicacid) were synthesized by a published procedure. Tetra(tert-butoxy)zirconium (TBZ) was distilled at 10⁻¹ torr and 80° C. The distilledproduct was stored in a nitrogen dry box, in the dark, and at −40° C.until needed. Otherwise, solvents were used as purchased. Titanium (0.25mm; 99.6%), aluminum (0.25 mm; 99.0%); and iron (0.125 mm; 99.5%) foilsand titanium Ti-6Al-4V billet (all obtained from Goodfellow, Inc.) wereprepared for use by sanding, followed by cleaning with methanol, and cutinto ca. 1 cm×1 cm coupons (foils) or 1 inch diameter disks (billet).The coupons were dried for at least an hour before use, and stored in anoven at 200° C.

The first set of comparative examples demonstrate the films which can beformed on various native metal oxide surfaces using ambient temperaturecontact of the surface with a carboxylic and a phosphonic acid, both ofwhich represent classes of art-recognized oxide surface derivatizingagents.

COMPARATIVE EXAMPLES Comparative Example 1 Carboxylic Acid Treatment ofAluminum Native Oxide

A coupon of aluminum was prepared as described above. A 1.0 mM solutionof stearic acid in iso-octane was prepared for deposition on thealuminum coupon. Deposition was carried out by immersing the aluminumcoupon into the stearic acid solution for 24 hours, then washed withfresh iso-octane. The presence of a stearic acid film was confirmed byIR spectroscopy. The self-assembled monolayer alignments were confirmedby contact angle measurements. Washing the substrates after they wereimmersed in the carboxylic acid solutions aided in the removal ofmolecules that were not bound to the aluminum, but were merely sittingon the surface.

The films formed in solution were stable. The stearic acid film, whichformed in 24 hours, was removed by anhydrous ethyl ether under mildconditions in the same amount of time. The monolayer-coated aluminumsubstrate was placed in ether at room temperature without using anystirring device. Removal of a significant portion of the film within 90minutes was confirmed by IR spectroscopy. After removing the monolayer,it was possible to establish another monolayer on the aluminum surfaceby repeating the same technique. This could be done repeatedly, butthere was a gradual erosion of the aluminum substrate.

From the IR information, it was apparent that the interaction betweenthe carboxylic acid and the metal oxide substrate surface was weak, asillustrated by the ability to produce and remove the monolayer undermild conditions. The nature of the interaction is apparently hydrogenbonding between the acid and the hydroxyls on the surface of the metal.Apparently, covalent bonds are not formed because, if they were, muchmore vigorous conditions would be required to remove the carboxylic acidfrom the surface of the metal oxide.

Comparative Examples 2-4 Ambient Phosphonic Acid Treatment of Aluminum,Iron, and Titanium Native Oxide Surfaces

Samples of coupons of aluminum, iron, and titanium, prepared asdescribed above, were treated with an aerosol of n-octadecanephosphonicacid in tetrahydrofuron (THF) at room temperature in the ambientenvironment. Following the spray application of the acid solution thesolvent was allowed to evaporate at ambient temperature and thederivatized surfaces of the coupons were analyzed by FTIR. The surfaceswhere then washed with THF and analyzed both before and after a peeltest using red Scotch® “650” tape. The analysis shows that on iron, thephosphonic acid forms a layer on the native surface oxide that, while ofsparse coverage, survives both washing and peel testing. In the case ofthe aluminum samples, a weakly bound phosphonic acid layer is formedthat survives washing, but not peel testing. For the titanium sample,any phosphonic acid which absorbed to the surface was readily removed bywashing with the deposition solvent.

Comparative Examples 5 Vacuum-Anneling of Phosphonic Acid CoatingApplied to Titanium Native Oxide Surfaces

A titanium coupon, prepared as described above was treated with a 0.8 mMTHF solution of octadecanephosphonic acid in the form of an aerosolspray under dry N₂. The treated coupon was placed under vacuum (10⁻¹torr), and sealed off. The coupon remained in the evacuated vessel forsix hours. DRIFT analysis before and after rinsing of the sampledemonstrated that none of the phosphonic acid remained on the surfaceafter rinsing in THF.

The next group of examples demonstrates derivatization of titanium oxidesurfaces according to the present invention using a phosphonic acid andphosphonic acid derivatives.

Example 1 Formation of a Bound Phosphonic Acid Film on a Titanium NativeOxide Surface

A titanium coupon, prepared as described above was treated with a 0.6 mMTHF solution of octadecanephosphonic acid in the form of an aerosolspray under dry N₂. The treated coupon was left under a current of drynitrogen until the solvent evaporated. Following solvent evaporation thesample was heated for 18 hours at 110° C. in air. The coupon was cooledto ambient temperature and rinsed twice with THF. This cycle ofapplication, heat annealing, and rinsing was repeated five times. DRIFTanalysis of the resulting coating on the coupon demonstrated that aphosphonate surface coating was bonded to the surface and remained afterrinsing and peel testing. The coupons thus prepared were stored in anoven at 200° C. there being no upper limit on annealing time.

Example 2 Formation of a Difunctional, Bound Phosphonic Acid Film on aTitanium Native Oxide Surface

A 5 mM solution of 11-phosphonoundecanoic acid in dry, distilled THF wasaerosol sprayed onto a titanium coupon prepared as described above usingthe procedure described above for preparation of films usingdifunctional phosphonic acids. Analysis by infrared spectroscopy (IR) ofthe resulting surface films produced show the characteristic IRstretches observed for alkyl chains and for bound phosphonic acids,indicating that the phosphonate group was bound to the surface of thecoupon and the ω-carboxylic acid groups were oriented away from thesurface and hydrogen bonded.

Example 3 Formation of a Difunctional, Bound Hydroxy-Phosphonic AcidFilm on a Titanium Native Oxide Surface

A 10 mM THF solution of 11-hydroxyundecylphosphonic acid was applied toa titanium coupon, prepared as described above, as an aerosol using theprocedure described for Example 2, except that baking of the sample waslimited to 18 hours post application. Infrared analysis indicated thatthe phosphonic acid portion of the coating precursor was bound to thenative oxide and showed a broad peak between 3300-3600 cm⁻¹ indicativeof hydrogen bonded hydroxyl groups as well as characteristic peaks forthe aliphatic chain.

Example 4 Formation of Bound, Mixed-Difunctional Phosphonic Acid Coatingon a Titanium Native Oxide Surface

Using the procedure described for Example 2, above, coatings comprisingmixtures of 11-phosphonoundecanate acid and either 4-phosphonobutyrate,decanephosphonate or a mixture of these species in any ratio will beprepared by aerosol applications of a solution containing a mixture ofthese species. The ratio of surface bound materials will be found to beclose to that of the ratio of acid constituents of the solution applied.Subsequent coupling chemistry (with, for example, a phenol or an aminoacid) can be accomplished to optimize yields of elaborated surface filmsby controlling the microenvironments of the ω-carboxlic acid termini inthis manner. Similar experiments can be done for mixtures containingω-hydroxyalkylphosphonate as well.

In the second and third groups of examples, following, films formed ontitanium metal coupons using difunctional phosphonic acids (both theωcarboxylic acid and co-hydroxyl functional films) are further reactedwith moieties useful in demonstrating the reactivity of the layer andwith other moieties which are useful in the promotion of bone adhesion.

Examples 5-6 Further Derivatization of Titanium Oxide Surface Boundω-Difunctional 11-Undecanoic Phosphonic Acid

In this second group of examples, the free carboxylic acid portion ofthe difunctional phosphonate layer applied to the surface of a titaniumcoupon prepared according to Example 2 is further reacted at thecarboxylic acid site by esterification of the acid with a phenol, anamino-acid, and with a peptide.

Example 5 Formation of Amino Acid Amides of Bound ω-difunctional11-Undecanoic Phosphonic Acid Coating on a Titanium Native Oxide Surface

Coupons were derivatized with a carbodiimide/hydroxysuccinimide couplingreagent. Coupons prepared according to Example 2 were stirred in anaqueous solution (75 mM) of1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimide and 20 mMN-hydroxysuccinimide to form the imido adduct of the acid. The couponsthus treated were then transferred to a beaker of a 75 mM solution oflysine in water. The coupons were then extensively rinsed with water anddried under vacuum. FTIR analysis indicates the presence of an amide(coupling of the carboxylic acid of the phosphonate and the aminefunctional group of the lysine amino acid).

Example 6 Formation of Peptide Thio-esters of Bound ω-difunctional11-Undecanoic Phosphonic Acid Coating on a Titanium Native Oxide Surface

The use of a surface of the present invention to bind short peptides toa Ti or alloy surface is demonstrated next. A cyateine-modified peptide,RGDC (Arg-Gly-Asp-Cys), described above which occurs in Firbronectincell-adhesive protein, is selected for attachment to the carboxylic acidends of surface layers on titanium coupons prepared according to Example2, above. The coupons are first treated with a solution ofdicyclohexylcarbodiimide and N-(6-hydroxyhexyl)maleimide indichloromethane to provide the co-carboxylic acid groups of thederivatized surface of the coupon with a maleimide ester group that willreact with thiol functionality in the cysteine of the modified peptide.Foils are then bathed in an aqueous solution of RGDC (Arg-Gly-Asp-Cys)to couple the thiol group of cysteine to the immobilized maleimidegroup, leading to the attachment of RGDC to the co-carboxylicacid-maleimide, yielding a bioactivated Ti surface.

In this second group of examples, the free hydroxide portion of thedifunctional phosphonate layer applied to the surface of a titaniumcoupon prepared according to Example 3 using11-hydroxy-undecylphosphonic acid is further reacted at the hydroxyfunctional group by conventional organic chemistry with dansyl chloride,an amino-acid, and with a peptide

Example 7 Reaction of Titanium Oxide Surface Bound

ω-Difunctional 11-Hydroxy-Undecyl Phosphonic Acid with Dansyl Chloride

Titanium coupons prepared according to Example 3 above were placed in asolution of approximately 10 mg of dansyl chloride and 0.1 mL oftriethylamine in 10mL of acetonitrile. The solution was stirred for 18hours under N₂. Coupons were removed from the solution, blotted dry andrinsed with acetonitrile. The coupons were then subjected to FTIRanalysis which indicates, by the presence of new peaks at 1600 cm⁻¹ andat 1200-1100 cm⁻¹, that a sulfonate ester formed. The dansyl adduct hasa characteristic flourescence spectrum, and fluorescence microscopicanalysis of the coupons confirms the formation of the surface-bounddansyl ester product. The fluorescence spectroscopy also indicates thatthe coating is dense-coverage and uniform over the entire surface of thecoupon.

For the next two examples, the surface bound hydroxyphosphonate isconverted into the maleimide adduct according to the followingprocedure. A coupon prepared according to Example 3, above, is placedinto a solution of about 15 mg of3-maleimido-(propionic-acid-N-hydroxysuccinimide) ester in 10 mL ofacetonitrile. The coupon is then transferred into a nitrogen glove boxand place into an ambient temperature maleimide solution for 24-72 hrs,after which, coupons are removed from the solution and blotted dry, andrinsed with acetonitrile. FTIR analysis indicates peaks corresponding to11-hydroxyundecylphosphonate bound to the surface of the coupon throughthe phosphonate functional group, with additional peaks at 1731 and 1707cm⁻¹ corresponding to the carbonyl stretches of the maleimide adduct.

Examples 8 Reaction of Titanium Oxide

Surface Bound ω-Difunctional 11-Hydroxy-Undecyl Phosphonic Acid withCysteine

Coupons having the phosphonate-maleimide adduct, prepared as describedabove on titanium coupons prepared according to Example 3, were placedin a stirred solution of 15 mg. of cysteine dissolved in 10 mL of doublydistilled, Millipore™-filtered water for 8-24 hrs. The foils wereremoved from solution, dried, and rinsed in doubly distilled water. FTIRanalysis of the coupons showed changes in the maleimide carbonyl regionand a new peak at ca. 1700 cm⁻¹, indicative of the presence of cysteinebound to the coupon.

Examples 9 Reaction of Titanium Oxide Surface

Bound ω-Difunctional 11-Hydroxy-Undecyl Phosphonic Acid with a Peptide

Coupons containing the phosphoric coated-maleimide adduct, prepared asdescribed above from titanium coupons prepared according to Example 3,are placed in a solution of the peptide RGDC (Arg-Gly-Asp-Cys) used toprepare coupons of Example 7, above. The RGDC solution comprises about10 mg of the peptide in 10 mL of doubly distilled, Millipore™-filteredwater. Coupons are stirred at ambient temperature for about 8 to about48 hours. The coupons are removed from solution, dried, and rinsed indoubly distilled water. FTIR, analysis before and after peptidetreatment demonstrates changes in the meleimide carbonyl region andbroadening in the carboxylate region (˜700 cm⁻¹) which persists aftertwo solvent rinses, indicating the presence of the RGDC tetrapeptidebound to the coupons.

It will be appreciated that peptides and amino acids can be “tagged”with a fluorescent marker by covalent bonding a small fluourescentspecies, such as dansyl chloride as an ester or thioester to the parentcompound. It will be appreciated that amino acids and peptides which arebound to phosphonate species bonded to oxide surfaces, such as aredescribed above can be tagged before or after such bonding reactions.When the surface species amino acid and peptide adducts are “tagged” inthis manner, examination of the coupons by fluorescence microscopy afterderivitization indicates the coatings of the peptide bounded to thecoupon are dense and uniform over the entire coupon.

The next group of examples demonstrates the dense-coverage of a titaniumnative oxide surface that can be achieved with the coating of thepresent invention.

Examples 10-11 Formations of a Phosphonate Coating on a Titanium QuartzMicrobalance Electrode

As described above, quartz microbance electrodes were treated withoctylphosphonic acid and 11 hydroxy undecylphosphonic acid to form anoctylphosphonate and undecylphosphonate coating on the native oxidelayer on the electrodes, the results of microbalance measurement of thedensity of surface coverage for the two species is presented below inTable I.

TABLE 1 Surface coverage of Ti by alkylphosphonates. Phosphonate Δ f(Hz)Coverage (nmol/cm²)^(a,b) Octylphosphonate 109 2.46 (1.89) 115 2.60(2.0) 11-Hydroxyundecylphosphonate 141 2.46 (1.89) 165 2.87 (2.21) 1152.00 (2.21 140 2.44 (1.88) ^(a)As measured by QCM ^(b)Corrected valuefor surface roughness factor measured to be 1.3 by AFM analysis of thesputtered Ti electrode given in ( ).

Example 12 Atomic Force Microscopy of an Octadecylphosphonate Coating

A coating of octadecylphosphonate on the native oxide surface of apolished titanium coupon was prepared by the aerosol method describedabove using a 0.75 mM THF solution of octadecylphosphonic acid. The acidsolution was applied under nitrogen and evaporated using the ambientmethod. The spray-heat-rinse cycle was repeated 6 times. The resultantcoating was studied by atomic force microscopy (AFM) using a Dimension3000 (Digital Instruments) operated in “soft” TappingMode. An AFMmicrograph of the polished Ti foil surface shows it to have grooves(resulting from the polishing process), but regions between thesegrooves are appreciably flat (mean roughness≈0.7 nm; FIG. 3). Sectionanalyses examined surface roughness in more detail. The morphology ofthe surface changed dramatically on formation of the octadecylphosphonicacid film. On coated coupons, AFM micrograph and section analysis showedislands (typical diameter≈50 nm) of similar height (≈2.2 nm), consistentwith monolayer formation on the surface, and the mean roughness of thesurface increased to 1.5 nm on monolayer attachment. With reference tofilm height data obtained for a self-assembled monolayer of this samephosphonic acid on mica (≈1.8 nm), an alkyl chain tilt angle of about33° was estimated. The AFM analysis indicates the coating is ofdense-coverage. Correction of the microbalance results of Examples 10and 11 by the AFM data indicate a surface coverage in excess of 20 timesof that observed for reactions of the native oxide hydroxyl sites,described above.

The next example demonstrates the use of a functionalizedalkyl-phosphonate coating of the present invention to bond a bone-growthpromoting peptide RGDC (described above) to the surface of a titaniumalloy (Ti-6Al-4V), and the use of this derivatized surface in adheringand proliferating osteoblasts.

Example 13 Application of a Phosphonate Coating to a Titanium Alloy andSubsequent Peptide Derivatization to Provide an Osteoblast AdhesionPromoting Surface

Disks of Titanium Alloy Ti-6Al-4V prepared as described above werecoated with a layer of 11-hydroxyundecylphosphonate by placing them in avessel filled with a 10 mM THF solution of 11-hydroxyundecylphosphonicacid. The THF was allowed to evaporate and the disks were then baked inan oven at 120° C. for 48 hours and were rinsed in dry THF. Thus preparethe titanium alloy disks were further derivatized with the cysteinemodified fibronectin cell attachment peptide argynine glycine asparticacid (RGDC) which has been described above.

The RGDC peptide [American Peptide] was bonded to the phosphonatecoating using a maleimide coupling procedure. The maleimide derivativeof the hydroxy functionalized phosphonate coating was prepared byplacing the coated disk into a 5 mM acetonitrile solution of3-maleimidopropionic acid-N-hydroxysuccinimide ester for a period of 24hours at room temperature. Thus prepared the maleimide adduct was rinsedwith a fresh acetonitrile solution. The disks were transferred into anacetonitrile solution of the peptide described above, RGDC, withstirring for 24 hours, yielding the peptide bound via a thiol esterlinkage through the cysteine residue to the hydroxy end of thephosphonate coating.

These modified titanium alloy disks were examined for their interactionwith human osteoblasts. Human fetal osteoblasts (HFOB 1.19; ATCC) weremaintained in a 1:1 mixture of Ham's F12 and Dulbecco's modified Eagle'smedium (DMEM), without phenol red (GIBCO, BRL), 10% fetal bovine serum(Hyclone Laboratories) and 0.3 mg/ml G418 (GIBCO, BRL). Cells werelabeled with 10 μM Cell Tracker Orange (Molecular Probes, Oreg.) for 30minutes at 34° C. After this time, the medium was removed and replacedwith fresh medium and serum for an additional 30 minutes at 34° C. Cellswere released from tissue culture dishes using 0.2 mg/ml EDTA in PBS,washed with PBS, resuspended in serum-free medium at 1×10⁵/ml, and 500μl of the cell suspension was added to wells containing the metal disksubstrates which had been blocked with 1% BSA in PBS for 30 minutesbefore cell addition. Cells were allowed to spread on the substrates for90 minutes, after which time they are washed with PBS and visualizedusing a Nikon Optiphot-2 microscope. Images were captured using aPhotometrics Coolsnap camera and analyzed using Coolsnap and IP labssoftware.

The results of this study indicate that human osteoblasts can adhere andpropogate on surfaces prepared according to the present invention.

Examples 1-12 were duplicated by treating coupons made from titaniumalloy Ti-6Al-4V (Goodfellows) under the same conditions and with thesame reagents used for the titanium coupons used in Examples 1-12.Results were the same, demonstrating that the coatings of the presentinvention can be applied equally well to the native oxide of titaniumalloys using the methods of the present invention.

The next group of examples demonstrates the use of phosphoric acid toform an intermediate layer on titanium metal native oxide surfaces whichmay be further derivatized with other moieties, and a derivatizedsurface which can promote osteoblast adhesion and spreading.

Examples 14-15 Dip-Treatment of Titanium Native Oxide Surfaces inPhosphoric Acid Solution

In Example 14, a coupon of Titanium foil (99.6+% annealed), prepared asdescribed above, was immersed in 1.4M aqueous H₃PO₄ (pH=1.5) at roomtemperature for one hour, then heated at 110° C. for greater than 16hours. After two rinsings with THF, examination by DRIFTS showed that ithad a coating of Ti(H₂PO₄)₃ (hereinafter Ti-phosphate) remained thatcould not be rinsed away.

In Example 15, titanium coupons prepared and described above were dippedin an aqueous solution of phosphoric acid (1.45 m; pH 1.5) for 1 hr atambient temperature an pressure. The coupons were then removed fromsolution and warmed in an oven at 120° C. for 18 hrs., then cooled,rinsed with water, and “peeled” with masking tape to remove any weaklyadsorbed material. X-ray powder diffraction analysis and DiffuseReflectance Fourier Transform Infrared analysis (DRIFT) confirmed thepresence of phosphate coating (Ti-phosphate).

Coated coupons were prepared in accordance with Example 15 and furtherderivitized using the spray/heat/rinse procedure described above usingthe reagents indiated below in Table II.

TABLE II Solute Baking concentration/ temperature/ Example Derivatizingspecies solvent time 16 octadecyl(triethoxy) 1.8 mM/THF 120° C./24 hrs.silane 17 octadecanethiol 1.0 mM/THF  60° C./24 hrs. 18 octadecylamine1.0 mM/THF  60° C./18 hrs. 19 octadecyl(triethoxy)- 0.8 mM/THF 120°C./16 hrs. silane

In each case, adherent, dense-coverage coatings of the reactant found onthe surface of the phosphate coated coupon by IR analysis.

Example 20 Treatment of Phosphate Coatings With Hydrolytically ReactiveMegal Alkoxides

Coupons prepared according to Example 15 were put in a horizontal tubewhich could be externally cooled and which was equipped with twostopcocks for exposure to vacuum (10⁻³ Torr) or to vapor phasetetra(tert-butoxy)zirconium (Zr(O-t-but)₄). Coupons were subjected tothree cycles each consisting of alternating exposure to vapor ofZr(O-t-but)₄ with external evacuation for 15 min, followed by 30 minexposure to the organometallic reagent vapor without externalevacuation. The first cycle was done at room temperature, and the lattertwo with external cooling by dry ice. Coupons were then subjected toroom temperature vacuum (10⁻³Torr) for 16 hrs to remove any physisorbedZr(O-t-but)₄. DRIFT analysis confirmed formation of dense-coveragesurface Zr alkoxide.

Example 21 Derivatization of a Dense-Coverage Zr Alkoxide Bound toTitanium

Coupons prepared according to Example 20 were sprayed with 1.75 mMsolution of octadecylphosphonic acid in dry tetrahydrofuran (THF).Samples were evacuated overnight (0.1 Torr), rinsed with THF, “peeled,”tested and analyzed by DRIFT. The analysis demonstrated an adherentalkyl-phosphonate coating bonded to the zirconated surface.

The derivitization reactions of Examples 14 through 21 were repeated,using the same reagents and conditions on coupons of Ti-6Al-4V(Goodfellows) prepared according to the procedure described for Example15 above. Analysis of the filing showed that titanium alloy can bederivitized in the same manner with the same results seen from thetitanium.

The next group of examples demonstrates the use of a phosphate coatingof the present development to provide a derivatized surface on atitanium material which promotes the adhesion and proliferation ofosteoblasts.

Examples 22-23 Adhesion of Osteoblasts To a Titanium Material PhosphateCoated Peptide Derivatized Surface

Disks cut from titanium billet and from titanium alloy Ti-6Al-4V billetwere prepared and coated with a phosphate coating according to Example15.

A phosphate coated disk of each material was placed in a Teflon® welland they were each treated with a solution of amino propyl-(triethoxy)-silane (10 mM in THF), and then solvent rinsed, with sonication,to give surface-bound 3-amino-propyl siloxanes. These disks were thenfurther derivatized by placing each in a 5 mM acetonitrile solution of3-maleimidopropionic-acid-N-hydroxysuccinimide-ester for 18 hours atroom temperature to give the maleimide adduct. The disks were removedfrom solution, solvent evaporated, and analyzed by IR. They were thenrinsed in acetonitrile, with sonication, and dried in vacuo (0.1 Torr).The disks were further derivatized by placing them into a solution ofthe RGDC peptide used in Example 13 above, (5 mM), prepared in 5 ml ofpurified water (Millipore), with the pH adjusted to 6.5 using 0.1M NaOH.The disks remained in the peptide solution stirring at room temperaturefor 24 hours. Formation of the surface bound RGDC was verified by IR.The disks were then rinsed with water, dried, subjected to tape peeltesting, and reanalyzed by IR. The peptide coating was found to beadherent.

The peptide coated disks were subjected to the human osteoblast testdescribed above in Example 13. The results showed that the surfacepromoted the adhesion and proliferation of osteoblasts.

Example 24 Derivitazation of Phosphate Coating with11-Mercaptoundecanoic Acid

Mercaptoundecanoic acid was recrystallized from ethanol at roomtemperature. A solution of mercaptoundecanoic acid (1.0 mM in THF) wasapplied by aerosol deposit onto coupons of titanium and of titaniumalloy Ti-6Al-4V. The coupons were placed under N₂ for 6-12 hrs in ahorizontal tube equipped with a stopcock to regulate N₂ flow andpressure, then evacuated at 0.1 Torr for at least 4 hrs, and analyzed byDRIFT. A dense coating of the mercaptan was found adhered to the surfaceof both the metal and alloy.

1. An adherent, self-assembled phosphorous-based coating layer bonded tothe native oxide surface of a material by heating a self-assembledphosphorous-based acid layer formed on the surface thereof until saidlayer is bonded thereto, wherein said phosphorous-based acid is selectedfrom the group consisting of phosphoric acid and organophosphonic acids.2. The coating layer of claim 1 formed on the native oxide surface of atitanium material substrate.
 3. The coating layer of claim 2, whereinsaid phosphorous-based acid is phosphoric acid.
 4. The coating layer ofclaim 2, wherein said phosphorous-based acid is an organo-phosphonicacid containing a hydrocarbon ligand having from about 2 to 40 carbonatoms, said hydrocarbon ligand comprising a linear or branched,saturated or unsaturated, substituted or unsubstituted aliphatic oraromatic alkylene moiety.
 5. The coating layer of claim 4, wherein saidhydrocarbon ligand is a saturated or unsaturated, substituted orunsubstituted alkyl group
 6. The coating layer of claim 4, wherein saidhydrocarbon ligand is substituted by an aromatic substituent.
 7. Thecoating layer of claim 4 wherein said hydrocarbon contains a ligandcomprising an aromatic moiety.
 8. The coating layer of claim 4, whereinsaid hydrocarbon ligands are omega-substituted.
 9. The coating layer ofclaim 4, wherein said hydrocarbon ligands are substituted at one or morecarbon positions.
 10. The coating layer of claim 8, wherein saidomega-substituent is selected from the group consisting of hydroxyl,amino, carboxylate and thiol groups.
 11. The coating layer of claim 1,wherein said native oxide surface comprises a titanium oxide surfacebonded to a material selected from the group consisting of metal, metaloxide, ceramic, and polymers.
 12. The coating layer of claim 2, whereinsaid titanium material is a titanium alloy.
 13. The coating layer ofclaim 12, wherein said titanium alloy is alloy Ti-6Al-4V.
 14. A methodof bonding a layer of a phosphorous-based acid moiety to a titaniumoxide surface comprising a coating said oxide surface with aphosphorous-based acid moiety self-assembled layer and heating saidcoated oxide surface until said self-assembled layer is bonded thereto,wherein said phosphorous-based acid is selected from the groupconsisting of phosphoric acid and organo-phosphonic acids.
 15. Themethod of claim 14, wherein the titanium oxide surface is the nativeoxide of a titanium material substrate.
 16. The method of claim 14wherein said oxide surface layer is heated to a temperature betweenabout 20 and about 200° C.
 17. The method of claim 14, wherein saidphosphorous-based acid moiety is an unsaturated organo-phosphonic acidcontaining a hydrocarbon ligand having from about 2 to 40 carbon atoms,said hydrocarbon ligand comprising a linear or branched, saturated orunsaturated, aliphatic or aromatic alkylene moiety.
 18. The method ofclaim 17, wherein said hydrocarbon ligand comprises an unsaturatedalkylene moiety, and the method further comprises the step ofpolymerizing said unsaturated alkylene moiety.
 19. The method of claim17, wherein said organo-phosphate hydrocarbon ligand contains api-electron delocalized structure.
 20. The method of claim 19, whereinsaid pi-electron delocalized structure is an aromatic ring compound. 21.The method of claim 14 wherein the phosphorous-based acid is phosphoricacid, thereby forming a hydroxyl-containing phosphate coating on saidoxide surface.
 22. The method of claim 21 further comprising the step ofreacting hydroxyl groups resident on said phosphate coating with a metalalkoxide reagent having two or more alkoxide ligands, thereby forming alayer comprising said metal alkoxide covalently bonded to said phosphatecoating, said metal alkoxide layer comprising unreacted alkoxideligands.
 23. The method of claim 22, wherein said metal alkoxide isselected from the group consisting of reagents comprising metals fromgroups 4-14 ligated with 2 or more alkoxide ligands.
 24. The method ofclaim 23, wherein said metal alkoxide has the formula M(tert-butoxide)₄,where M is Zr or Si.
 25. The method of claim 24, wherein said titaniumoxide surface is a derivative surface of a polymeric, metallic, orceramic material with a surface bearing a titanium oxide layer.
 26. Animplantable device having one or more surfaces for attachment to bonetissue, wherein at least one attachment surface comprises the coatedsubstrate of claim
 1. 27. An implantable device having one or moresurfaces for attachment to bone tissue, wherein at least one attachmentsurface comprises the coated substrate of claim
 2. 28. The implantabledevice of claim 27, comprising a knee or hip replacement joint.
 29. Themethod of claim 21, further comprising the step of bonding to saidhydroxyl groups, moieties selected from the group consisting of moietiesfor the covalent attachment of bone tissue proteins and the chemicalprecursors thereof.
 30. The method of claim 29, wherein said moietycomprises a thiol or amido substituent.
 31. The method of claim 14wherein said step of coating said surface with a self-assembled layercomprises: a) coating said surface with a solution of saidphosphorous-based acid; and a) evaporating the solvent from saidsolution coating.
 32. The method of claim 31, wherein said acidcomprises an organo-phosphonic acid having a hydrocarbon ligandcontaining from 2 to 40 carbon atoms.
 33. The method of claim 32,wherein said hydrocarbon ligand is a saturated or unsaturated,substituted or unsubstituted, aliphatic or aromatic alkylene group. 34.The method of claim 33, wherein said hydrocarbon ligand isomega-substituted with a substituent selected form the group consistingof hydroxyl, amino, carboxylate or thiol groups.
 35. The implantabledevice of claim 27, wherein said coated surface of said implantcomprises titanium or an alloy thereof.
 36. A method for attaching animplantable device to bone tissue in a patient in need thereofcomprising implanting in said patient the device of claim
 27. 37. Amethod for improving the adhesion to bone tissue of implantable medicaldevices having a titanium oxide surface for contacting said bone tissue,said method comprising coating said surface with phosphoric acid or aphosphonic acid according to the method of claim
 14. 38. A method forattaching an implantable device to bone tissue, said device having atitanium oxide surface for contacting said bone tissue, said methodcomprising first coating said oxide surface with a phosphorous-basedacid moiety according to the method of claim
 14. 39. A method forcoating an implantable device for attachment to bone tissue, said devicehaving a titanium oxide surface for contacting said bone tissue, saidmethod comprising coating said surface with phosphoric- or anorgano-phosphonic acid according to the method of claim
 31. 40. A coateddevice produced by the method of claim
 37. 41. The coated device ofclaim 40, wherein said surface is coated with an organo-phosphonic acidhaving an organic ligand containing from about 2 to 40 carbon atoms. 42.The coated device of claim 41, wherein said organic ligand is asaturated or unsaturated, substituted or unsubstituted, aliphatic oraromatic alkylene moiety.
 43. The coated device produced by the methodof claim 40, wherein said surface is coated with phosphoric acid,thereby providing an inorganic phosphate coating comprising freehydroxyl groups.
 44. The coated device of claim 43, further comprisingthe step of derivatizing said free hydroxyl groups by covalentattachment of a moiety selected from the group consisting of bone tissueproteins and the chemical precursors thereof.
 45. The coated device ofclaim 44, wherein said chemical precursors comprise thiol substituents.46. The device of claim 44, comprising a knee or hip replacement joint.47. The device of claim 46, wherein said titanium oxide surfacecomprises the native oxide surface of titanium or an alloy thereof. 48.A method for attaching an implantable device to bone tissue in a patientin need thereof comprising implanting in said patient the device ofclaim 40.